FACS-based mCherry-BFP competition assay

DCK*-IKZF1 293FT cells were infected with a mixture of two lentiviruses encoding Cas9 and either (i) sgDCK and mCherry or (ii) sgCTRL and BFP (blue fluorescent protein). The two lentiviruses were mixed together such that the ratio of mCherry-positive to BFP-positive cells after infection and puromycin selection was 1:99. An analogous experiment was set up using a lentivirus encoding sgCTRL and mCherry. The pool of infected cells was plated at 20,000 cells per well in a six-well plate and then cultured in media containing either 100 μM BVdU or DMSO for 21 days. Cells were collected for FACS analysis on days 1, 6, 18, and 33. After each time point, cells were reseeded at 20,000 cells per well and treated with fresh BVdU.

Jurkat cells that had been stably infected to express Cas9 and DCK*-FOXP3 were superinfected with lentivirus encoding either (i) sgDCK and mCherry or (ii) sgCTRL and BFP. These cells were mixed together and analyzed by FACS to achieve a final ratio of mCherry-positive to BFP-positive cells of 1:99. The pool of infected cells was plated at 40,000 cells/ml in a six-well plate and then cultured in media containing either 100 μM BVdU or DMSO for 14 days. Cells were collected for FACS analysis on days 6 and 14.

The Jurkat cells expressing Cas9 and ASCL1-DCK* that were used for the CRISPR-Cas9 screen described above were superinfected with lentiviruses encoding sgRNAs targeting CDK2, ASCL1, or a nontargeting sgRNA as a control, the fluorescent protein mCherry and a puromycin resistance gene or with a lentivirus encoding a nontargeting sgRNA as a control, and the fluorescent protein BFP and a puromycin resistance gene (see schema in fig. S16F). The cells were selected with puromycin. mCherry puromycin-resistant cells were then mixed with BFP puromycin-resistant cells at a 1:3 ratio as determined by FACS analysis. The mixed cells were plated at 5 × 10⁴ cells/ml and then cultured in media containing 500 μM BVdU or DMSO (0) for 18 days. FACS analysis was performed every 6 days. After each FACS analysis, fresh BVdU was added, and the density of the cells was adjusted to 5 × 10⁴ cells/ml with fresh media.